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1.
2.
Subtypes of purinergic receptors involved in modulation of cytoplasmic calcium ion concentration ([Ca2+]i) and insulin release in mouse pancreatic β-cells were examined in two systems, pancreatic islets in primary culture and beta-TC6
insulinoma cells. Both systems exhibited some physiological responses such as acetylcholine-stimulated [Ca2+]i rise via cytoplasmic Ca2+ mobilization. Addition of ATP, ADP, and 2-MeSADP (each 100 μM) transiently increased [Ca2+]i in single islets cultured in the presence of 5.5 mM (normal) glucose. The potent P2Y1 receptor agonist 2-MeSADP reduced insulin secretion significantly in islets cultured in the presence of high glucose (16.7 mM),
whereas a slight stimulation occurred at 5.5 mM glucose. The selective P2Y6 receptor agonist UDP (200 μM) transiently increased [Ca2+]i and reduced insulin secretion at high glucose, whereas the P2Y2/4 receptor agonist UTP and adenosine receptor agonist NECA were inactive. [Ca2+]i transients induced by 2-MeSADP and UDP were antagonized by suramin (100 μM), U73122 (2 μM, PLC inhibitor), and 2-APB (10
or 30 μM, IP3 receptor antagonist), but neither by staurosporine (1 μM, PKC inhibitor) nor depletion of extracellular Ca2+. The effect of 2-MeSADP on [Ca2+]i was also significantly inhibited by MRS2500, a P2Y1 receptor antagonist. These results suggested that P2Y1 and P2Y6 receptor subtypes are involved in Ca2+ mobilization from intracellular stores and insulin release in mouse islets. In beta-TC6 cells, ATP, ADP, 2-MeSADP, and UDP
transiently elevated [Ca2+]i and slightly decreased insulin secretion at normal glucose, while UTP and NECA were inactive. RT-PCR analysis detected mRNAs
of P2Y1 and P2Y6, but not P2Y2 and P2Y4 receptors. 相似文献
3.
The mechanism of translocation of UDP-GlcNAc, UDP-Gal and UDP-Glc into intracellular vesicles has been studied using thymocytes whose plasma membranes have been permeabilized with isotonic ammonium chloride. It has been previously shown that the intracellular vesicles have specific carriers for UDP-GlcNAc and UDP-Gal. We now report that the translocation of these two sugar nucleotides occurs via UDP-GlcNAc/UDP and UDP-Gal/UDP antiports. The entry of UDP-GlcNAc or UDP-Gal into vesicles was specifically dependent on the exit of UDP from these vesicles. In contrast, no antiport mechanism has been recovered with UDP-Glc for which no transport and accumulation into intracellular vesicles were observed. 相似文献
4.
Noronha-Matos JB Costa MA Magalhães-Cardoso MT Ferreirinha F Pelletier J Freitas R Neves JM Sévigny J Correia-de-Sá P 《Journal of cellular physiology》2012,227(6):2694-2709
This study aimed at investigating the expression and function of uracil nucleotide‐sensitive receptors (P2Y2, P2Y4, and P2Y6) on osteogenic differentiation of human bone marrow stromal cells (BMSCs) in culture. Bone marrow specimens were obtained from postmenopausal female patients (68 ± 5 years old, n = 18) undergoing total hip arthroplasty. UTP and UDP (100 µM) facilitated osteogenic differentiation of the cells measured as increases in alkaline phosphatase (ALP) activity, without affecting cell proliferation. Uracil nucleotides concentration‐dependently increased [Ca2+]i in BMSCs; their effects became less evident with time (7 > 21 days) of the cells in culture. Selective activation of P2Y6 receptors with the stable UDP analog, PSB 0474, mimicked the effects of both UTP and UDP, whereas UTPγS was devoid of effect. Selective blockade of P2Y6 receptors with MRS 2578 prevented [Ca2+]i rises and osteogenic differentiation caused by UDP at all culture time points. BMSCs are immunoreactive against P2Y2, P2Y4, and P2Y6 receptors. While the expression of P2Y6 receptors remained fairly constant (7~21 days), P2Y2 and P2Y4 became evident only in less proliferative and more differentiated cultures (7 < 21 days). The rate of extracellular UTP and UDP inactivation was higher in less proliferative and more differentiated cell populations. Immunoreactivity against NTPDase1, ‐2, and ‐3 rises as cells differentiate (7 < 21 days). Data show that uracil nucleotides are important regulators of osteogenic cells differentiation predominantly through the activation of UDP‐sensitive P2Y6 receptors coupled to increases in [Ca2+]i. Endogenous actions of uracil nucleotides may be balanced through specific NTPDases determining whether osteoblast progenitors are driven into proliferation or differentiation. J. Cell. Physiol. 227: 2694–2709, 2012. © 2011 Wiley Periodicals, Inc. 相似文献
5.
Flores RV Hernández-Pérez MG Aquino E Garrad RC Weisman GA Gonzalez FA 《Molecular and cellular biochemistry》2005,280(1-2):35-45
Purification of HA-tagged P2Y2 receptors from transfected human 1321N1 astrocytoma cells yielded a protein with a molecular size determined by SDS-PAGE
to be in the range of 57–76 kDa, which is typical of membrane glycoproteins with heterogeneous complex glycosylation. The
protein phosphatase inhibitor, okadaic acid, attenuated the recovery of receptor activity from the agonist-induced desensitized
state, suggesting a role for P2Y2 receptor phosphorylation in desensitization. Isolation of HA-tagged P2Y2 nucleotide receptors from metabolically [32P]-labelled cells indicated a (3.8 ± 0.2)-fold increase in the [32P]-content of the receptor after 15 min of treatment with 100 μM UTP, as compared to immunoprecipitated receptors from untreated
control cells. Receptor sequestration studies indicated that ∼40% of the surface receptors were internalized after a 15-min
stimulation with 100 μM UTP. Point mutation of three potential GRK and PKC phosphorylation sites in the third intracellular
loop and C-terminal tail of the P2Y2 receptor (namely, S243A, T344A, and S356A) extinguished agonist-induced receptor phosphorylation, caused a marked reduction
in the efficacy of UTP to desensitize P2Y2 receptor signalling to intracellular calcium mobilization, and impaired agonist-induced receptor internalization. Activation
of PKC isoforms with phorbol 12-myristate 13-acetate that caused heterologous receptor desensitization did not increase the
level of P2Y2 receptor phosphorylation. Our results indicate a role for receptor phosphorylation by phorbol-insensitive protein kinases
in agonist-induced desensitization of the P2Y2 nucleotide receptor. (Mol Cell Biochem xxx: 35–45, 2005) 相似文献
6.
Patrizia Rubini Javorina Milosevic Johannes Engelhardt Mahmoud Al-Khrasani Heike Franke Attilla Heinrich Beata Sperlagh Sigrid C. Schwarz Johannes Schwarz Wolfgang Nörenberg Peter Illes 《Cell calcium》2009,45(5):485-498
Nucleotides play an important role in brain development and may exert their action via ligand-gated cationic channels or G protein-coupled receptors. Patch-clamp measurements indicated that in contrast to AMPA, ATP did not induce membrane currents in human midbrain derived neuronal progenitor cells (hmNPCs). Various nucleotide agonists concentration-dependently increased [Ca2+]i as measured by the Fura-2 method, with the rank order of potency ATP > ADP > UTP > UDP. A Ca2+-free external medium moderately decreased, whereas a depletion of the intracellular Ca2+ storage sites by cyclopiazonic acid markedly depressed the [Ca2+]i transients induced by either ATP or UTP. Further, the P2Y1 receptor antagonistic PPADS and MRS 2179, as well as the nucleotide catalyzing enzyme apyrase, allmost abolished the effects of these two nucleotides. However, the P2Y1,2,12 antagonistic suramin only slightly blocked the action of ATP, but strongly inhibited that of UTP. In agreement with this finding, UTP evoked the release of ATP from hmNPCs in a suramin-, but not PPADS-sensitive manner. Immunocytochemistry indicated the co-localization of P2Y1,2,4-immunoreactivities (IR) with nestin-IR at these cells. In conclusion, UTP may induce the release of ATP from hmNPCs via P2Y2 receptor-activation and thereby causes [Ca2+]i transients by stimulating a P2Y1-like receptor. 相似文献
7.
Biased agonism describes a multistate model of G protein-coupled receptor activation in which each ligand induces a unique structural conformation of the receptor, such that the receptor couples differentially to G proteins and other intracellular proteins. P2Y receptors are G protein-coupled receptors that are activated by endogenous nucleotides, such as adenosine 5′-triphosphate (ATP) and uridine 5′-triphosphate (UTP). A previous report suggested that UTP may be a biased agonist at the human P2Y11 receptor, as it increased cytosolic [Ca2+], but did not induce accumulation of inositol phosphates, whereas ATP did both. The mechanism of action of UTP was unclear, so the aim of this study was to characterise the interaction of UTP with the P2Y11 receptor in greater detail. Intracellular Ca2+ was monitored in 1321N1 cells stably expressing human P2Y11 receptors using the Ca2+-sensitive fluorescent indicator, fluo-4. ATP evoked a rapid, concentration-dependent rise in intracellular Ca2+, but surprisingly, even high concentrations of UTP were ineffective. In contrast, UTP was slightly, but significantly more potent than ATP in evoking a rise in intracellular Ca2+ in 1321N1 cells stably expressing the human P2Y2 receptor, with no difference in the maximum response. Thus, the lack of response to UTP at hP2Y11 receptors was not due to a problem with the UTP solution. Furthermore, coapplying a high concentration of UTP with ATP did not inhibit the response to ATP. Thus, contrary to a previous report, we find no evidence for an agonist action of UTP at the human P2Y11 receptor, nor does UTP act as an antagonist. 相似文献
8.
Nucleoside di- and triphosphates and adenosine regulate several components of the mucocilairy clearance process (MCC) that
protects the lung against infections, via activation of epithelial purinergic receptors. However, assessing the contribution
of individual nucleotides to MCC functions remains difficult due to the complexity of the mechanisms of nucleotide release
and metabolism. Enzymatic activities involved in the metabolism of extracellular nucleotides include ecto-ATPases and secreted
nucleoside diphosphokinase (NDPK) and adenyl kinase, but potent and selective inhibitors of these activities are sparse. In
the present study, we discovered that ebselen markedly reduced NDPK activity while having negligible effect on ecto-ATPase
and adenyl kinase activities. Addition of radiotracer [γ
32P]ATP to human bronchial epithelial (HBE) cells resulted in rapid and robust accumulation of [32P]-inorganic phosphate (32Pi). Inclusion of UDP in the incubation medium resulted in conversion of [γ
32P]ATP to [32P]UTP, while inclusion of AMP resulted in conversion of [γ
32P]ATP to [32P]ADP. Ebselen markedly reduced [32P]UTP formation but displayed negligible effect on 32Pi or [32P]ADP accumulations. Incubation of HBE cells with unlabeled UTP and ADP resulted in robust ebselen-sensitive formation of
ATP (IC50 = 6.9 ± 2 μM). This NDPK activity was largely recovered in HBE cell secretions and supernatants from lung epithelial A549
cells. Kinetic analysis of NDPK activity indicated that ebselen reduced the V
max of the reaction (K
i = 7.6 ± 3 μM), having negligible effect on K
M values. Our study demonstrates that ebselen is a potent non-competitive inhibitor of extracellular NDPK. 相似文献
9.
An existing enzymic method for preparing [γ-32P]ATP from 32Pi has been modified toyield [γ-32P]ATP free of salt and buffer. 32P is incorporated into the γ-position of ATP by isotopic exchange in the presence of glyceraldehyde 3-phosphate dehydrogenase and 3-phosphoglycerate kinase. Unreacted 32Pi is separated from [γ-32P]ATP by column chromatography on Dowex 1 bicarbonate. [γ-32P]ATP is eluted with 2 m triethylammonium bicarbonate, which is then completely removed by freeze-drying. 相似文献
10.
A rapid method for the measurement of [γ-32P]ATP specific radioactivity in tissue extracts containing other 32P-labeled compounds is described. The neutralized acid extract is incubated with cyclic AMP-dependent protein kinase, cyclic AMP and casein. The incorporation of 32P into casein from [γ-32P]ATP is measured by perchloric acid precipitation of the protein on filter paper. 32P-Casein formation is linearly related to the specific radioactivity of the [γ-32P]ATP. Separation of ATP from other 32P-labeled compounds is not required for the assay. Application of this method in the evaluation of [γ-32P]ATP specific radioactivity in two rat cardiac muscle preparations exposed to 32Pi is demonstrated. 相似文献
11.
After conversion of unlabeled DNA and RNA to 3′-mononucleotides accurate base compositional analysis can be performed on as little as 10 ng of the hydrolysate. The 3′-mononucleotides are first quantitatively postlabeled with [γ-32P]ATP by T4 polynucleotide kinase and are then separated as mononucleoside diphosphates on Whatman DE-81 ion-exchange paper at pH 3.5 after hydrolysis of surplus [γ-32P]ATP to 32P1. The locations of the four labeled nucleoside diphosphates are determined by autoradiography and the ratio of radioactivity in the four spots gives the base ratio of the sample. 相似文献
12.
(i) A new, rapid method for the measurement of [γ-32P]ATP specific radioactivity in tissue extracts in the presence of other 32P-containing compounds is described. The deproteinized extract is incubated with phosphorylase b and phosphorylase kinase, and the incorporation of 32P into protein from [γ-32P]ATP is measured by precipitation on filter paper in trichloroacetic acid. No separation of ATP or other treatment of the extracts is required for the assay. (ii) 32Pi uptake in perfused rat heart was found to be a relatively slow process, with a Km of 0.084 mm, whereas equilibration between intracellular 32Pi and [γ-32P]ATP occurred rapidly. 相似文献
13.
Preparation of (beta-32P)ribonucleoside-5'-triposphates using permeable cells of Escherichia coli 总被引:1,自引:0,他引:1
A rapid method for the preparation of [β-32P]ribonucleoside-5′-triphosphates is described. The method involves the incubation of a ribonucleoside triphosphate with 32Pi and E. coli cells made permeable to nucleotides. The labeled triphosphates can be isolated by preparative thin layer chromatography on poly(ethylene)imine cellulose plates. Labeled GTP, CTP, and UTP obtained by this method are more than 99% pure [β-32P]compounds. Labeled ATP contains about equal amounts of label in the β- and γ-phosphate position. Pure [β-32P]ATP can be obtained from this preparation by exchanging the γ-32P against unlabeled Pi and reisolating the labeled ATP by charcoal adsorption and elution. 相似文献
14.
Mechanisms of agonist-dependent and -independent desensitization of a recombinant P2Y2 nucleotide receptor 总被引:3,自引:0,他引:3
Otero M Garrad RC Velázquez B Hernández-Pérez MG Camden JM Erb L Clarke LL Turner JT Weisman GA González FA 《Molecular and cellular biochemistry》2000,205(1-2):115-123
UTP activates P2Y2 receptors in both 1321N1 cell transfectants expressing the P2Y2 receptor and human HT-29 epithelial cells expressing endogenous P2Y2 receptors with an EC50 of 0.2- 1.0 M. Pretreatment of these cells with UTP diminished the effectiveness of a second dose of UTP (the IC50 for UTP-induced receptor desensitization was 0.3 - 1.0 M for both systems). Desensitization and down-regulation of the P2Y2 nucleotide receptor may limit the effectiveness of UTP as a therapeutic agent. The present studies investigated the phenomenon of P2Y2 receptor desensitization in human 1321N1 astrocytoma cells expressing recombinant wild type and C-terminal truncation mutants of the P2Y,2 receptor. In these cells, potent P2Y2 receptor desensitization was observed after a 5 min exposure to UTP. Full receptor responsiveness returned 5-10 min after removal of UTP. Thapsigargin, an inhibitor of Ca2+-ATPase in the endoplasmic reticulum, induced an increase in the intracellular free calcium concentration, [Ca2+]i, after addition of desensitizing concentrations of UTP, indicating that P2Y2 receptor desensitization is not due to depletion of calcium from intracellular stores. Single cell measurements of increases in [Ca2+]i induced by UTP in 1321N1 cell transfectants expressing the P2Y2 receptor indicate that time- and UTP concentration-dependent desensitization occurred uniformly across a cell population. Other results suggest that P2Y2 receptor phosphorylation/dephosphorylation regulate receptor desensitization/resensitization. A 5 min preincubation of 1321N1 cell transfectants with the protein kinase C activator, phorbol 12-myristate 13-acetate (PMA), reduced the subsequent response to UTP by about 50% whereas co-incubation of PMA with UTP caused a greater inhibition in the response. The protein phosphatases - 1 and -2A inhibitor, okadaic acid, partially blocked resensitization of the receptor. Furthermore, C-terminal truncation mutants of the P2Y2 receptor that eliminated several potential phosphorylation sites including two for PKC were resistant to UTP-, but not phorbol ester-induced desensitization. Down regulation of protein kinase C isoforms prevented phorbol ester-induced desensitization but had no effect on agonist-induced desensitization of wild type or truncation mutant receptors. These results suggest that phosphorylation of the C-terminus of the P2Y2 receptor by protein kinases other than protein kinase C mediates agonist-induced receptor desensitization. A better understanding of the molecular mechanisms of P2Y2 nucleotide receptor desensitization may help optimize a promising cystic fibrosis pharmacotherapy based on the activation of anion secretion in airway epithelial cells by P2Y2 receptor agonists. 相似文献
15.
Jacob M. Dundee Mar Puigdellívol Richard Butler Thomas O. J. Cockram Guy C. Brown 《Aging cell》2023,22(2)
Aging causes loss of brain synapses and memory, and microglial phagocytosis of synapses may contribute to this loss. Stressed neurons can release the nucleotide UTP, which is rapidly converted into UDP, that in turn activates the P2Y6 receptor (P2Y6R) on the surface of microglia, inducing microglial phagocytosis of neurons. However, whether the activation of P2Y6R affects microglial phagocytosis of synapses is unknown. We show here that inactivation of P2Y6R decreases microglial phagocytosis of isolated synapses (synaptosomes) and synaptic loss in neuronal–glial co‐cultures. In vivo, wild‐type mice aged from 4 to 17 months exhibited reduced synaptic density in cortical and hippocampal regions, which correlated with increased internalization of synaptic material within microglia. However, this aging‐induced synaptic loss and internalization were absent in P2Y6R knockout mice, and these mice also lacked any aging‐induced memory loss. Thus, P2Y6R appears to mediate aging‐induced loss of synapses and memory by increasing microglial phagocytosis of synapses. Consequently, blocking P2Y6R has the potential to prevent age‐associated memory impairment. 相似文献
16.
17.
In this study, we examined the response of glioma C6 cells to 2′,3′-O-(4-benzoylbenzoyl)-ATP (BzATP) and showed that the BzATP-induced calcium signaling does not involve the P2X7 receptor activity. We show here that in the absence of extracellular Ca2+, BzATP-generated increase in [Ca2+]i
via Ca2+ release from intracellular stores. In the presence of calcium ions, BzATP established a biphasic Ca2+ response, in a manner typical for P2Y receptors. Brilliant Blue G, a selective antagonist of the rat P2X7 receptor, did not reduce any of the two components of the Ca2+ response elicited by BzATP. Periodate-oxidized ATP blocked not only BzATP- but also UTP-induced Ca2+ elevation. Moreover, BzATP did not open large transmembrane pores. What is more, a cross-desensitization between UTP and
BzATP occurred, which clearly shows that in glioma C6 cells BzATP activates most likely the P2Y2 but not the P2X7 receptors. 相似文献
18.
A satisfactory method for the determination of the specific activity of highly labeled [γ-32P]ATP has not been reported previously. Yields of high specific activity 32P labeled material usually are too small to be detected by ultraviolet spectrophotometry or phosphate analysis. Recent reports describing the assay of ATP by enzyme catalyzed phosphate transfer to 3H labeled glucose (1) or galactose (2) are not suitable for use with highly labeled 32P material since the crossover into the 3H channel will greatly exceed the radioactivity of the 3H labeled phosphate acceptor. Recently Schendel and Wells reported the preparation of essentially carrier free [γ-32P]ATP. They indicated, however, that the specific activity of the labeled product could not be determined by conventional methods (3). We have developed and now routinely use an expedient method for the determination of the specific activity of picomole quantities of highly labeled [γ-32P]ATP. This procedure measures the phosphate transfer from [γ-32P]ATP to oligothymidylic acid [dT(pT)10] catalyzed by bacteriophage T4 induced polynucleotide kinase. The specific activity is determined by measuring the radioactivity present in d-32pT(pT)10, and can be verified by an isotope dilution method employing the same assay. Specific activities as high as 240 Ci/mmole have been determined. 相似文献
19.
Assay of adenosine 3', 5' cyclic monophosphate by stimulation of protein kinase: a method not involving radioactivity 总被引:2,自引:0,他引:2
In order to meet a need for a cAMP assay which is not subject to interference by compounds in plant extracts, and which is suitable for use on occasions separated by many 32P half-lives, an assay based on cAMP-dependent protein kinase has been developed which does not require the use of [γ-32P]ATP. Instead of measuring the cAMP-stimulated increase in the rate of transfer of [γ-32P] phosphate from [γ-32P]ATP to protein, the rate of loss of ATP from the reaction mixture is determined. The ATP remaining after the protein kinase reaction is assayed by ATP-dependent chemiluminescence of the firefly luciferin-luciferase system. Under conditions of the protein kinase reaction in which a readily measurable decrease in ATP concentration occurs, the logarithm of the concentration of ATP decreases in proportion to the cAMP concentration, i.e., the reaction can be described by the equation: [ATP] = [ATP]0 e?[cAMP]kt. The assay based on this relationship can detect less than 1 pmol of cAMP. The levels of cAMP found with this assay after partial purification of the cAMP from rat tissue, algal cells, and the media in which the cells were grown agreed with measurements made by the cAMP binding-competition assay of Gilman, and the protein kinase stimulation assay based on transfer of [32P] phosphate from [γ-32P]ATP to protein. All of the enzymes and chemicals required for the assay of cAMP by protein kinase catalyzed loss of ATP can be stored frozen for months, making the assay suitable for occasional use. 相似文献
20.
GTPase from rod outer segments: characterization by photoaffinity labeling and tryptic peptide mapping 总被引:2,自引:0,他引:2
D J Takemoto B E Haley J Hansen O Pinkett L J Takemoto 《Biochemical and biophysical research communications》1981,102(1):341-347
The photoaffinity label [γ-32P]8-N3GTP has been used to identify GTP-binding components in highly purified preparations of GTPase from bovine rod outer segments. These preparations contain two major polypeptides of 37,000 and 39,000 daltons. In the presence of photolyzing radiation, [γ-32P]8-N3GTP is covalently attached to the 37,000 dalton polypeptide. Tryptic peptide mapping of this polypeptide indicates that it is highly related to the 39,000 dalton species that has been previously identified as a GTP-binding component. 相似文献